Preparation method of lactoferrin-polyphenol nanocomposite with intestinal immune function

Abstract

This invention discloses a method for preparing lactoferrin-polyphenol nanocomposites with intestinal immune function. The method involves the following steps: First, lactoferrin is moderately hydrolyzed under the action of a complex protease to optimize its molecular structure. Then, it is compounded with water-soluble polyphenols (such as chlorogenic acid or epicatechin) using metal ion chelation technology, and Ca²⁺ is used to induce lactoferrin bridging and aggregation to form a complex. Next, ultrasonic homogenization is performed to further control the particle size, ensuring it is distributed within the range of 100-300 nm. The resulting complex is then concentrated by nanofiltration and electrostatic spray drying to obtain a structurally stable shell-core nanocomposite, which is then nitrogen-filled to prevent oxidation. The lactoferrin-polyphenol nanocomposite prepared by this invention not only exhibits good stability and intestinal immune-enhancing effects but also significant antibacterial activity, making it widely applicable in functional foods and nutritional supplements.

Description

Technical Field

[0001] This invention relates to the field of food nutrition and functional factors, specifically to a lactoferrin-polyphenol nanocomposite with intestinal immune function, its preparation method, and its application. Background Technology

[0002] Lactoferrin is a multifunctional protein that is naturally found in milk. Due to its antibacterial, antiviral, anti-inflammatory, and immunomodulatory biological activities, it is widely used in the food, health products, and pharmaceutical fields.

[0003] However, lactoferrin suffers from a series of technical limitations in practical applications, which restrict its bioactivity and stability, especially during processing and storage.

[0004] First, lactoferrin is susceptible to enzymatic hydrolysis, pH and temperature changes. It is prone to degradation under high temperature or acidic conditions, leading to a decrease in activity.

[0005] Secondly, lactoferrin molecules are relatively large and do not easily cross the intestinal barrier, affecting the absorption and utilization of its immune-active components in the human body.

[0006] In addition, lactoferrin itself has a high positive charge, which makes it easy to form large-particle aggregates when it binds with polyphenolic compounds, which is not conducive to the formation of stable nanocomposite structures.

[0007] The aforementioned issues not only affect the interaction between lactoferrin and polyphenols, but also reduce the dispersibility and stability of the nanocomposite in the intestine, thereby limiting its bioactivity. Summary of the Invention

[0008] The technical problem to be solved by the present invention is to overcome the shortcomings of the prior art and provide a lactoferrin-polyphenol nanocomposite that protects the activity of lactoferrin while significantly enhancing its intestinal immune function, as well as its preparation method, and propose an application for the lactoferrin-polyphenol nanocomposite.

[0009] The technical solution of this invention: This invention discloses a method for preparing lactoferrin-polyphenol nanocomposites with intestinal immune function, comprising the following steps:

[0010] S1. Lactoferrin hydrolysis;

[0011] S2. The formation of lactoferrin-polyphenol complex is induced by metal ion chelation technology. Water-soluble polyphenolic compounds are added to the lactoferrin hydrolysate, followed by calcium chloride. Ca2+ is used to induce the negatively charged lactoferrin to bridge and aggregate, and encapsulate the polyphenolic compounds in the aqueous phase.

[0012] S3. Ultrasonic homogenization treatment induces the structural deconstruction and reconstruction of the lactoferrin-polyphenol complex;

[0013] S4. Nanofiltration concentration and polarized spray drying.

[0014] Furthermore, in step S1, the lactoferrin hydrolysis includes moderate hydrolysis of lactoferrin using a combination of endopeptidases such as papain, bromelain, and gingerase. The amount of compound protease added is 2000-3000 U / g, the hydrolysis pH is 6.5-8.5, the hydrolysis temperature is 50-65℃, the hydrolysis time is 2-4 h, and the ratio of any two proteases is 2:1-1:2.

[0015] Furthermore, in step S2, under pH 4-8 conditions, water-soluble polyphenolic compounds such as chlorogenic acid are added to the lactoferrin hydrolysate obtained in step S1, and then 0.1mM-0.5mM calcium chloride is added to induce the negatively charged lactoferrin to aggregate and encapsulate the chlorogenic acid and other polyphenolic compounds in the aqueous phase.

[0016] Furthermore, in step S3, the ultrasonic treatment power is 30-60Hz and the treatment time is 5-15 minutes. After ultrasonic treatment, the size of the lactoferrin-polyphenol nanocomposite shrinks from 10-100μm to 100-300nm and is stably dispersed.

[0017] Furthermore, in step S4, an electrostatic spray drying device is used to further induce the lactoferrin-polyphenol nanocomposite to undergo structural remodeling with different charged micro-bends, forming a stepwise microstructure with the polar head facing inward and the non-polar head facing outward, and forming a shell-core encapsulation structure for water-soluble polyphenols.

[0018] Furthermore, the inlet air temperature for spray drying is 90-100℃, and the outlet air temperature is 40-50℃.

[0019] Furthermore, after step S4, the lactoferrin-polyphenol nanocomposite is also nitrogen-filled and packaged.

[0020] Furthermore, in step S1, the lactoferrin has a purity greater than 80%.

[0021] This invention also discloses a lactoferrin-polyphenol nanocomposite with intestinal immune function. The composite is formed by the chelation of lactoferrin and water-soluble polyphenols with metal ions, has a particle size of 100-300 nm, possesses a shell-core structure, and exhibits enhanced intestinal immune function and antibacterial activity.

[0022] This invention also discloses an application of a lactoferrin-polyphenol nanocomposite with intestinal immune function. The lactoferrin-polyphenol nanocomposite prepared based on the above-mentioned method for preparing lactoferrin-polyphenol nanocomposite with intestinal immune function, or based on the above-mentioned lactoferrin-polyphenol nanocomposite, can enhance intestinal immune function by stimulating intestinal epithelial cells to secrete immune factors.

[0023] The beneficial effects of this invention compared to the prior art are as follows:

[0024] By optimizing enzymatic hydrolysis conditions, introducing metal ion chelation technology, and employing ultrasonic homogenization, the stability and bioavailability of the complex were effectively improved. This invention's technical solution significantly enhances the intestinal immune function while protecting the activity of lactoferrin, thus possessing significant application value.

[0025] This invention systematically optimizes the hydrolysis conditions (such as enzyme combination, pH, temperature, and time to achieve moderate hydrolysis) and chelation process (pH value and calcium ion concentration) of lactoferrin, thereby ensuring its successful binding with polyphenols to form a stable nanocomplex while preserving its bioactivity. This optimization not only avoids excessive damage to the lactoferrin structure but also enhances the activity and structural stability of the complex, making the product suitable for long-term storage and various functional applications. Attached Figure Description

[0026] Figure 1 This is a diagram showing the relative molecular mass distribution of the lactoferrin hydrolysate products in this invention.

[0027] Figure 2 This is the intrinsic fluorescence spectrum of lactoferrin and its polyphenol complex at 298 K in this invention;

[0028] Figure 3 This refers to the average particle size of lactoferrin and its polyphenol complex in this invention;

[0029] Figure 4 This refers to the average particle size change of the lactoferrin-polyphenol complex after ultrasound in this invention.

[0030] Figure 5 This invention illustrates the effect of spray drying on the polyphenol retention rate of the lactoferrin-polyphenol complex.

[0031] Figure 6 This invention relates to the effect of lactoferrin-polyphenol complex on the expression of cellular immune factors.

[0032] Figure 7 This invention relates to the effect of lactoferrin-polyphenol complex on the Th1 / Th2 differentiation balance of intestinal cells. Detailed Implementation

[0033] To enhance understanding of the present invention, we will now describe it in further detail with reference to the accompanying drawings. These embodiments are for illustrative purposes only and do not constitute a limitation on the scope of protection of the present invention.

[0034] This invention provides a method for preparing lactoferrin-polyphenol nanocomposites with intestinal immune function. The method includes five steps: moderate hydrolysis of lactoferrin, induction of complexation using metal ion chelation technology, deconstruction and reconstruction of the polyphenol nanocomposite structure, nanofiltration concentration, and polarized spray drying.

[0035] 1. Raw material selection and pretreatment

[0036] The lactoferrin raw material used in this invention is commercially available or laboratory-isolated lactoferrin with a purity greater than 80%, ensuring the quality and activity of the lactoferrin. The polyphenols used are water-soluble chlorogenic acid and epicatechin, which have good antioxidant and immunomodulatory activities.

[0037] 2. Lactoferrin hydrolysis

[0038] Objective: To moderately hydrolyze lactoferrin to enhance its structural stability while avoiding the formation of large-particle polymers in subsequent steps.

[0039] step:

[0040] Dissolve lactoferrin in deionized water and adjust the lactoferrin concentration to 5% (w / v). Add a complex protease (papain, bromelain, ginger protease) at a concentration of 2500 U / g lactoferrin.

[0041] Hydrolysis conditions:

[0042] pH: 7.5

[0043] Temperature: 55℃

[0044] Hydrolysis time: 3 hours

[0045] Enzyme ratio: Papain:Bromelain:Gingerase = 2:1:1

[0046] Experimental data:

[0047] The degree of hydrolysis of lactoferrin after hydrolysis was 58.60%. HPLC analysis showed that the molecular weight distribution of the hydrolyzed lactoferrin was stable and its structure was intact.

[0048] Control group: Under the same conditions, no compound enzyme was added, and lactoferrin maintained its original structure.

[0049] like Figure 1As shown, after hydrolysis by a complex enzyme, lactoferrin is mainly degraded into small peptide fragments with a molecular weight of less than 5000 Da, accounting for 61.25%, followed by hydrolyzed fragments of 500-5000 Da at 24.06%, and a small amount (14.69%) of hydrolyzed fragments >5000 Da. The molecular weight of protein hydrolysates is usually closely related to their biological activity. Studies have shown that peptides with a molecular weight between 500-1000 Da have high antioxidant and immunomodulatory activities, as well as good transdermal, hygroscopic, and moisturizing properties. These peptides are more easily absorbed and utilized by the human body than intact protein macromolecules. The molecular weight distribution results of this patent show that lactoferrin has a high content of 500-1000 Da digestion product fragments after hydrolysis by a complex enzyme. It is speculated that the complex enzyme hydrolysis increases the immunomodulatory activity of lactoferrin.

[0050] 3. Polyphenol non-covalent complexes

[0051] Objective: To induce lactoferrin and polyphenols to form a stable nanocomposite using Ca²⁺ chelation technology.

[0052] step:

[0053] The hydrolyzed lactoferrin solution was adjusted to pH 6.0, and water-soluble chlorogenic acid and epicatechin were added to achieve a polyphenol concentration of 20 mg / g lactoferrin. Subsequently, calcium chloride solution was added dropwise, with the Ca²⁺ concentration controlled at 0.3 mM, and the mixture was stirred thoroughly.

[0054] Experimental data:

[0055] Following the chelation reaction, the fluorescence intensity of the complex decreased significantly as measured by endogenous fluorescence spectroscopy, indicating that the polyphenols were successfully incorporated into the structure of lactoferrin. Particle size analysis showed that the average particle size of the complex was approximately 150 nm, and the particles were uniformly distributed.

[0056] Control group: No polyphenols and calcium chloride were added, and the process proceeded directly to the next step. The results showed that no stable complex was formed.

[0057] Endogenous fluorescence spectroscopy can reflect structural changes in proteins and their binding to molecules, and has become an effective technique for studying changes in the polar microenvironment after protein-polyphenol interactions.

[0058] like Figure 2 As shown, the fluorescence peak of the lactoferrin-polyphenol complex exhibited a significant red shift after the addition of 20 mg / g of water-soluble chlorogenic acid and epicatechin. This suggests that hydrogen bonding interactions may exist between lactoferrin and polyphenols within the hydrophobic cavities of the protein.

[0059] Furthermore, the fluorescence intensity of the lactoferrin-chlorogenic acid complex was significantly lower than that of the lactoferrin-epicatechin complex, indicating that the addition of chlorogenic acid leads to the unfolding of the lactoferrin structure and the exposure of more tryptophan and tyrosine groups compared to epicatechin. This suggests that chlorogenic acid has a higher binding affinity to lactoferrin than epicatechin.

[0060] Protein aggregation has a significant impact on protein solubility and other functional properties, and protein aggregation is typically assessed by characterizing protein particle size. Figure 3 Compared with lactoferrin, the particle size of the lactoferrin-polyphenol complex was significantly increased (P<0.05), indicating that lactoferrin interacted with chlorogenic acid and epicatechin to form a nanocomplex.

[0061] 4. Ultrasonic homogenization treatment of lactoferrin polyphenol nanocomposites

[0062] Objective: To further deconstruct and reconstruct the lactoferrin polyphenol complex through ultrasonic treatment to obtain a stable nanostructure.

[0063] step:

[0064] The chelated lactoferrin polyphenol complex was treated in an ultrasonic device with an ultrasonic power set at 45 Hz for 10 minutes. The temperature was controlled below 25℃ during the ultrasonic treatment.

[0065] Experimental data:

[0066] After ultrasonic treatment, the particle size of the complex decreased from 150 nm before chelation to 120 nm, and the complex exhibited a uniform dispersion in the solution.

[0067] Transmission electron microscopy (TEM) revealed that the complex was a regular spherical shape with a uniform distribution.

[0068] Control group: The untreated complex had larger particle size, uneven dispersion, and higher precipitation rate.

[0069] like Figure 4 As shown, the particle size of the lactoferrin-polyphenol complex was significantly reduced after ultrasonic treatment. This is because the cavitation-induced microfluidic effect can enhance molecular mass transfer and increase the collision frequency between lactoferrin and polyphenol molecules, thereby promoting the formation of smaller and more uniform complexes.

[0070] 5. Nanofiltration Concentration and Electrostatic Spray Drying

[0071] Objective: To increase the concentration of the complex and optimize the structure retention during spray drying.

[0072] step:

[0073] First, nanofiltration concentration was performed for 40 minutes, increasing the concentration of the lactoferrin polyphenol complex to 20%. The concentrated complex was then dried using an electrostatic spray dryer, with the inlet air temperature controlled at 95℃ and the outlet air temperature at 45℃.

[0074] Experimental data:

[0075] The dried lactoferrin-polyphenol complex powder exhibits a microscopic core-shell structure. Particle size analysis results show that the powder particle size after spray drying is approximately 150 nm, with a uniform particle size distribution. The dried complex demonstrates good stability, retaining 90% of the antioxidant activity of the polyphenols under storage conditions.

[0076] Control group: Conventional spray drying (without electrostatic treatment) was used. The results showed that the particle size of the complex was larger after drying, and the polyphenol activity was significantly lost.

[0077] 6. Nitrogen-filled packaging

[0078] Objective: To prevent oxidation and degradation of the complex during storage by using nitrogen-filled packaging.

[0079] step:

[0080] The dried lactoferrin polyphenol nanocomposite powder was placed in a nitrogen-filled packaging bag, sealed after nitrogen filling, and stored.

[0081] Experimental data:

[0082] After 3 months of storage, accelerated aging tests showed that the complex structure of the nitrogen-filled packaging group was intact and the polyphenol oxidation rate was less than 5%; the control group (without nitrogen filling) showed a polyphenol oxidation rate as high as 20%.

[0083] Feature Analysis

[0084] 1. Particle size and morphology analysis:

[0085] The particle size of the composite was determined using transmission electron microscopy (TEM) and dynamic light scattering. The results showed that the final composite particle size was in the range of 120-150 nm and exhibited a uniform spherical distribution.

[0086] 2. Stability Testing:

[0087] The accelerated aging test results showed that the composite powder had good dispersibility, no significant increase in particle size, and high stability after being stored at 40°C for 14 days.

[0088] 3. Bioactivity testing:

[0089] Intestinal immune function: In vitro cell experiments showed that the secretion of immune factors by intestinal epithelial cells treated with the complex was significantly increased.

[0090] In the complex immune response, IL-10, as a multifunctional cytokine, especially as B cell stimulating factor-2, can induce activated B cells to produce IgA. Immunocytokines, as key regulatory molecules of the immune system, not only regulate the activity and proliferation of immune cells but also coordinate the balance of various immune responses, and are often used as important indicators for assessing the strength of the body's immune function.

[0091] like Figure 3-6 As shown, the IL-10 levels in both the model group and the normal group were significantly lower than those in the normal group (P<0.05). This result reveals the successful establishment of the inflammatory cell model. Compared with the model group, the IL-10 level in the lactoferrin-polyphenol complex group was significantly increased (P<0.05), indicating that the lactoferrin-polyphenol complex can alleviate inflammation by upregulating IL-10 expression.

[0092] The differentiation balance of Th1 / Th2 cells is crucial for normal immune function, and its imbalance is closely related to the occurrence of various diseases. Using the Th1 / Th2 ratio as a balance indicator, this study further explored the effect of the lactoferrin-polyphenol complex on the Th1 / Th2 cytokine secretion balance of inflammatory intestinal cells. Figure 7 The results showed that, compared with the normal group, the Th1 / Th2 ratio in the model group was significantly increased (P<0.001), indicating Th1 overexpression in intestinal cells, which may be related to enhanced intestinal inflammatory response. Compared with the model group, the Th1 / Th2 ratio of the lactoferrin-polyphenol complex was significantly decreased (P<0.01). This indicates that the lactoferrin-polyphenol complex has immunomodulatory effects, and can repair the Th1 / Th2 imbalance by regulating cytokine secretion, thereby enhancing intestinal immune function and improving intestinal immune homeostasis.

[0093] Results Summary

[0094] This invention discloses a method for preparing lactoferrin-polyphenol nanocomposites with intestinal immune function. Through rational hydrolysis, polyphenol chelation, ultrasonic treatment, nanofiltration concentration, and electrostatic spray drying, effective compounding of lactoferrin and polyphenols is achieved, forming a structurally stable nanocomposite. Experimental results show that this nanocomposite exhibits significant intestinal immune function and antibacterial effect, and demonstrates good stability during storage.

[0095] In summary, this invention innovatively constructs a stable lactoferrin-polyphenol nanocomposite structure by combining metal ion chelation technology with ultrasonic homogenization. This method not only effectively solves the problem of easy aggregation of lactoferrin and polyphenols, but also improves the dispersibility and bioavailability of the complex in the intestine, making the active ingredients easier to be absorbed by the intestine and significantly enhancing the intestinal immune function of the product.

[0096] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the specific embodiments described above. The specific embodiments and descriptions in the specification are merely for further illustrating the principles of the invention. Various changes and modifications can be made to the present invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of the present invention is defined by the claims and their equivalents.

Claims

1. A method for preparing a lactoferrin-polyphenol nanocomposite with intestinal immune function, characterized in that, Includes the following steps: S1. Lactoferrin hydrolysis; wherein the lactoferrin purity is greater than 80%, and the lactoferrin hydrolysis is performed using a complex protease composed of papain, bromelain, and ginger protease to hydrolyze the lactoferrin. The amount of complex protease added is 2000-3000 U / g, the hydrolysis pH is 6.5 to 8.5, the hydrolysis temperature is 50 to 65℃, and the hydrolysis time is 2 to 4 hours. The enzyme ratio in the complex protease is: papain: bromelain: ginger protease = 2:1:

1. S2. The formation of a lactoferrin-polyphenol complex is induced using metal ion chelation technology. Water-soluble polyphenolic compounds are added to lactoferrin hydrolysate, followed by the addition of calcium chloride. 2+ The process induces the aggregation of negatively charged lactoferrin bridges and encapsulates polyphenolic compounds in the aqueous phase. Specifically, under pH 4-8 conditions, water-soluble chlorogenic acid and epicatechin are added to the lactoferrin hydrolysate obtained in step S1 to achieve a total polyphenol concentration of 20 mg / g lactoferrin. Then, 0.1 mM-0.5 mM calcium chloride is added, utilizing Ca... 2+ It induces the aggregation of negatively charged lactoferrin bridges and encapsulates polyphenolic compounds in the aqueous phase; S3. Ultrasonic homogenization treatment to induce the structural deconstruction and reconstruction of lactoferrin-polyphenol complex; the ultrasonic treatment parameters are 30-60Hz, the treatment time is 5-15 minutes, and the temperature is controlled below 25℃ during the ultrasonic treatment. After ultrasonic treatment, the size of lactoferrin-polyphenol nanocomplex shrinks from 10-100 μm to 100-300 nm and is stably dispersed. S4. Nanofiltration concentration and polarized spray drying; including using electrostatic spray drying equipment to further induce the lactoferrin-polyphenol nanocomposite to undergo structural rearrangement of different charged micro-bends, forming a microstructure distribution with polar heads facing inward and non-polar heads facing outward, forming a shell-core encapsulation structure for water-soluble polyphenols, and the spray drying inlet air temperature is 90-100℃ and the outlet air temperature is 40-50℃, and also includes nitrogen filling packaging of the lactoferrin-polyphenol nanocomposite.

2. A lactoferrin-polyphenol nanocomposite with intestinal immune function, characterized in that, The particles, prepared by the method described in claim 1, have a particle size of 100-300 nm and a shell-core structure.

3. The application of a lactoferrin-polyphenol nanocomposite with intestinal immune function, characterized in that, Based on the lactoferrin-polyphenol nanocomposite with intestinal immune function as described in claim 2, intestinal immune function is enhanced by stimulating intestinal epithelial cells to secrete immune factors.

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